T he response of chick intestine to vitamin D and its metabolites was studied in an organ culture preparation of chick ileum explants. Both 25-hydroxycholecalciferol (25-OHD₃) at a concentration of 20 ng/ml or greater and 1,25-dihydroxycholecalciferol [1,25-(OH)₂D₃] at a concentration of 50 pg/ml or greater stimulated the rate of accumulation of [³²P]phosphate and ⁴⁵Ca by the explants and the incorporation of [³H]thymidine into DNA. The accumulation of [³²P]phosphate by the explants was against a concentration gradient and inhibited by ouabain and dinitrophenol. Two saturable mechanisms appeared to mediate the cellular accumulation of phosphate with K_a of 0.0047 and 0.125 mM, respectively. The V_max of the lower affinity transport mechanism was accelerated by 1,25-(OH)₂D₃. Actinomycin D (5.0 μg/ml) did not block the intestinal response to 1,25-(OH)₂D₃ stimulation of both [³²p]phosphate and ⁴⁵Ca accumulation. Significant stimulation of [³²P]phosphate accumulation was observed 30 min after the addition of 1,25-(OH)₂D₃, preceding the sterol-induced increase in the rate of ⁴⁵Ca uptake by 30 min and the sterol-induced increase in [³H]thymidine incorporation into DNA by 150 min. Increasing extracellular phosphate concentration to 3.0 mM increased [³H]thymidine incorporation into DNA and the rate of ⁴⁵Ca uptake by the explants. Reducing extracellular phosphate concentration to 0.05 mM attenuated the response of the explants to 1,25-(OH)₂D₃. From these observations it is postulated that the primary action of vitamin D sterols in the intestine is to enhance the ability of the mucosal cell to accumulate phosphate. The data suggest that restoration of intracellular phosphate levels may then permit expression of the cells' response to vitamin D sterols.